1. Technical Field
The present invention relates to an optical imaging system using incoherent structured illumination, and more particularly, to a sub-diffraction scattered light imaging system using incoherent structured illumination.
2. Background
Fluorescent microscopy (FM) is known for its high sensitivity, high molecular discrimination, and simultaneous multicolor imaging capability, and is therefore a crucial tool for studying the fine structures of cells and organisms through fluorescent labeling. However, FM has two major weaknesses: the photo-bleaching of fluorescent dye and the insufficient spatial resolution of fluorescent images due to diffraction limit. Conventional wide-field FM provides a lateral resolution of from 220 to 300 nm and an axial resolution of from 800 to 1000 nm. Confocal laser scanning fluorescent microscopy increases the axial resolution from 400 to 500 nm and provides three dimensional (3D) sectioning images, but the increase of the lateral resolution is limited. In recent years, structured illumination microscopy (SIM) utilizes a structured light pattern to illuminate samples in order to break the diffraction limit to achieve a doubled resolution in fluorescence images.
As one application, three-dimensional structured illumination fluorescence microscopy (3D-SIFM) utilizes three diffracted laser beams to generate a 3D interference pattern for sample illumination, and accordingly transfers the high spatial frequency image data to be covered by the scope of the optical transfer function (OTF) of a wide-field microscope. The high frequency image data under irradiation at various pattern orientations are collected and processed by the image reconstruction algorithm to retrieve a high-resolution fluorescence image according to Gustafsson et al. 3D-SIFM is now able to provide twice as much resolution in both lateral and axial directions with true optical sectioning as compared to the conventional wide-field fluorescence microscopy.
SIM-based techniques are widely applied to the measurement of fluorescent light, but not frequently used in the measurement of scattered light from samples. Scattered light imaging can image transparent and label-free specimens of strong scattering in their native environment. The increasing importance of noble metal nanoparticles in biological and biomedical applications further makes scattered light imaging an attractive modality to investigate the behavior and interactions at the sub-cell level. Apart from having excellent biocompatibility and stability, noble metal nanoparticles feature a strong ability to scatter light and resistance to photobleaching.
The adoption of 3D-SIM to scattered light imaging is complicated. In 3D-SIFM, a coherent or partially coherent light source is used to generate a 3D interference pattern with high modulation contrast. Because the fluorescent light emitted from a sample is incoherent, studied equations and procedures for incoherent imaging can be followed to retrieve a high resolution image. In contrast, light scattered from a sample is a coherent process. With a coherent light source, the known mathematical modality for fluorescence image reconstruction is unsuitable. New coherent image retrieving procedures are required but the complexity in mathematical derivation prohibits advancement in the technology. Additionally, scattered light imaging suffers from the interference of reflected light generated at interfaces of different materials due to no emission filter to block the incident light. Although a dark-field scheme adds a mask to block the reflected light, this design unavoidably reduces the intensity of the scattered light and degrades the image resolution.
FIG. 1 shows a conventional optical system for 3D structured illumination 10. A coherent light beam is outputted by a coherent light source 11 and is received by a spatial light modulator (SLM) 12 positioned on the optical path created by the coherent light source 11. The SLM 12 then diffracts the single inputting light beam into a plurality of higher order beams, for example, 0, +1, and −1 order diffracted beams, as shown in FIG. 1. In order to converge the parallel diffracted beams, the convex lens L1 is positioned at a distance f1 from the SLM 12, wherein f1 is the focal length of the convex lens L1.
The parallel beams passing through lens L1 are converged and then diverged to enter convex lens L2. Lens L2 is positioned at a distance f2 from the convergent points and produces parallel beams which intersect each other, wherein the distance f2 is the focal length of the lens L2. The structured pattern is formed at a conjugate image plane 16, or a Fourier plane, at the distance f2 away from the lens L2. A convex lens L3 is positioned at a distance f3 away from the conjugate image plane 16, receiving the parallel beams and producing three converging beams toward objective lens Lobj. The depiction of the objective lens Lobj in FIG. 1 is rather a high-level presentation, practically a set of lenses are arranged in the objective 17. The distance between the objective lens Lobj and the lens L3 is the sum of f3 and fobj, wherein f3 is the focal length of the lens L3, and fobj is the focal length of the objective lens Lobj, respectively. In particular, f3 is the distance between the lens L3 and the back focal plane 16′ of the objective lens Lobj; whereas fobj is the distance between the objective lens Lobj and its back focal plane 16′. In the case where a set of lenses are arranged in the objective 17, the fobj is the effective focal length of the set of lenses. Three parallel beams passing through the objective lens Lobj then intersect each other at another image plane where a stage 15 accommodating a sample is positioned. The intersection of three parallel coherent beams produces a 3D structured pattern at the stage 15 where a sample having virtual thickness can be imaged in a sectioning fashion to reconstruct its 3D image.
FIG. 2 shows an optical imaging system 20 for 3D structured illumination. The combination of the coherent light source 21, the SLM 22, the stage 25, the objective 27, and a set of optical lenses (L1′, L2′, L3′, L4′, L5′, Lobj′) is substantially identical to the optical system shown in FIG. 1 with only a minor variation of the lens arrangement for the sake of experimental convenience. An adjustable mask 23 is positioned on or off the optical path to selectively filter the diffracted beams. The use of the mask 23 also adds versatility to the structured illumination optical system, for the system to operate in a wide-field (preserve only the 0 order diffracted beam) or a 2D structured pattern illumination (preserve only the +1 and −1 order diffracted beam) mode. The mask 23 depicted in FIG. 2 is positioned off the optical path, therefore no diffracted beam is blocked. A charge-coupled device (CCD) camera 24 is placed at a position suitable for receiving fluorescent light emitting from the sample, and a real space image can be reconstructed based on the signal received by the CCD camera 24.
The present invention is the first to combine 3D-SIM and scattered light imaging, successfully generating 3D incoherent structured illumination to avoid speckle scattering and complicated coherent image retrieval. A reflective light scattering microscope with 3D structured illumination (SI-RLSM) is disclosed in the present invention, and a lateral/axial resolution of 120 nm/430 nm is demonstrated based on the high-resolution SI-RLS image of 100 nm noble metal nanoparticles. The present invention can be applied to detect noble metal nanoparticles or strong scatters in biological specimens to provide high resolution and high contrast 3D scattered light images.